Human pluripotent stem cells (PSCs), which have the potential to form virtually any cell type in the body, have been the subject of intense research focus due to their potential application to regenerative medicine and the more recent potential for patient specific stem cells via reprogramming, allowing differentiated adult cells to be turned into pluripotent ones. However, in the shadow of this excitement, the potential to exploit pluripotent stem cells for fundamental studies in human developmental biology has been overlooked. This is especially pertinent given the fundamental physiological differences between the human and mouse - the closest relative where such studies are typically carried out. The long-term goal of my future research program is to use PSCs to discover and characterize new regulators (i.e. genes, metabolites, growth factors, non-coding RNA) of the human developmental processes. In order to accomplish this it will be necessary to create a platform (set of methods and analytical tools) to allow the modeling, tracing, and comparison of the paths by which human stem cells differentiate under natural and investigator-controlled situations. To this end, I will use a next-generation single-cell analysis instrumentation (CyTOF mass cytometry), which I have previously used to measure levels of protein expression, regulatory modifications, cell cycle and cell death across most known cell types in human bone marrow in response to drugs, cytokines, and growth factors, to characterize this system. I will first create a single cell template of pluripotent cells undergoing differentiation using standard differentiation conditions. I will then create a set of computational tools that will not only take into consideration the dynamic nature of cellular differentiation, but also enable the comparison of differentiating cell-types in different investigator controlled conditions. I will use stable gene knockdown approaches to target genes of interest based on proteins previously identified as differentially expressed in human PSCs versus their differentiated counterparts. This pipeline of experimentation will establis a new and unique platform (a method for data collection, a method for data analysis and interpretation, and a proof-ofconcept of it's implementation) for the characterizing putative regulators of cellular differentiation.